The NIEHS Sister Study recently identified a panel of five differentially methylated CpGs between breast cancer cases and control subjects using prospectively collected white blood cell (WBC) DNA and a genomic array of 27,578 CpG sites. The predictive power of WBC DNA methylation in these five CpG sites in the Sister Study exceeded that of the Gail model and GWAS single nucleotide polymorphisms. Our objective is to use prospectively collected blood samples to validate this methylation profile using highly targeted next-generation sequencing in women in the Prostate Lung Colorectal Ovarian Cancer (PLCO) Screening Trial who developed breast cancer and those who did not. We will further extend the findings of the Sister Study by capitalizing on serial bloods available in PLCO to robustly investigate the association in two different time intervals between WBC DNA collection and diagnosis (i.e., 1-2, 4-7 years). This analysis will provide the information necessary to distinguish whether WBC DNA methylation changes are a response to preclinical breast cancer and further clarify the lead time, and/or if they predict the future likelihood of breast cancer. Tis study has the potential to reduce mortality by leading to the development of a risk assessment tool for breast cancer. Characterization of a WBC DNA breast cancer-related risk profile could help in better targeting of risk-reduction strategies of high- risk women and could lead to the development of novel prevention strategies.